DESCRIPTION (adapted from applicant's description): The elongation phase of transcription by RNA polymerase II (RNAPII) is a key regulated step in the generation of mature mRNAs. Shortly after initiation, negative transcription elongation factors (N-TEFs) cause RNAPII to produce short, prematurely terminated transcripts. Productive elongation to generate long transcripts, which in higher eukaryotes may reach millions of nucleotides in length, requires the action of a positive transcription elongation factor (P-TEFb) that suppresses the effects of N-TEFs. The counterpoising activities of N-TEFs and P-TEFb thus regulate the fraction of initiating RNAPII molecules that produce full-length transcripts. The overall goal of the proposed research is to understand the biochemical mechanisms by which these factors affect RNAPII activity. Besides P-TEFb, three other factors, DSIF, NELF, and so-called Factor 2 recently were shown to be N-TEFs. Other positive and negative factors likely are involved as well. Aim 1 proposes to use a combination of in vitro and in vivo studies to define the cellular function of factor 2 in molecular detail. In particular, its role as a termination factor in mitosis and in interphase transcription will be elucidated. Preliminary results suggest that factor 2 may be cell cycle regulated and that it could play a key role in clearing transcription factors from DNA in preparation for chromosome condensation and segregation. In aim 2, a defined elongation control system will be used to define the properties of DSIF and NELF, to examine the role of the CTD in NELF and DSIF function, and to determine the precise target of P-TEFb. The system will also be used to analyze effects of other elongation factors on DSIF, NELF, and P-TEFb. In aim 3, it is proposed to compare transcription in the defined system to that in a crude system depleted of various factors. Additional factors that influence DSIF and NELF activity will be sought in fractionated extracts, and the roles of TFIIH and TatSF in elongation control will be examined.